CN101874107A - The purposes of glycoprotein VI (GPVI) inhibitor - Google Patents
The purposes of glycoprotein VI (GPVI) inhibitor Download PDFInfo
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- CN101874107A CN101874107A CN200880114053A CN200880114053A CN101874107A CN 101874107 A CN101874107 A CN 101874107A CN 200880114053 A CN200880114053 A CN 200880114053A CN 200880114053 A CN200880114053 A CN 200880114053A CN 101874107 A CN101874107 A CN 101874107A
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- gpvi
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- reperfusion injury
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- Medicinal Chemistry (AREA)
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- Peptides Or Proteins (AREA)
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Abstract
The present invention has put down in writing a kind of by using thrombocyte GPVI inhibitor to reduce the method for reperfusion injury and/or infraction.This method is used in heart attack or during the selectivity heart operation or treat sufferer afterwards.
Description
Related application
The application requires in the U. S. application No.60/984 of submission on October 31st, 2007,334, the right of priority of " Uses ofa Glycoprotein VI (GPVI) Inhibitor (purposes of Glycoprotein VI (GPVI) inhibitor) " is incorporated its full content into the application by reference.
Technical field
The present invention relates to suppress with platelet membrane glycoprotein VI (GPVI) inhibitor the method for reperfusion injury and/or infraction, described inhibitor comprises antibody, protein fragments and micromolecular compound.
Background technology
When to the coronary occlusion of heart blood supply, can cause heart attack.Usually owing to causing that arteries narrows down and closes, the formation of atherosclerosis and thrombus causes the generation of blocking.The shortage of blood supply is called ischemic.Cardiac muscle can only tolerate the oxygen starvation of short period of time and can in 20-120 minute infarct take place.Because the myocardial cell is ultimate differentiation to a great extent, the regenerative power of heart is extremely limited.Once there was the sufferer of heart attack all can carry a heart that has blocking tissue in its remaining years.Because the myocardium pump blood ability of infraction reduces, so these sufferers keep also can reducing the ability of health blood supply.After the heart attack, then congestive heart failure can take place, may also can cause heart trouble to be shown effect once more.The mobility of sufferer in heart failure weakens, quality of life descends and the lost of life.
According to the recent statistics of American Heart Association, only 1,200,000 example heart attack people such as (, Circulation, 113:e85-151,2006) Thom just there is every year in the U.S..At present the U.S. has 5,000, the sufferer of 000 heart failure and annual newly-increased 550,000 cases people such as (, Circulation, 113:e85-151,2006) Thom.
The coronary artery that blocks can treat and be got through again with angioplasty and/or thrombolytics, thereby the muscle of previous ischemic is poured into again.Though pour into again the muscle of treatment ischemic very necessary, contradiction be that perfusion itself also can produce extra damage to muscle again.To heart attack ideal methods of treatment is that myocardial infarction is minimized.Yet, since usually to heart attack be difficult to make a prediction, it is impossible that prophylactic treatment just becomes.Therefore, the treatment of angioplasty and/or thrombolytics may be more feasible with the combination that reduces reperfusion injury treatment (for example, using) in ambulance or first-aid room.The treatment that reduces reperfusion injury will promote to have a heart attack/recovery of ischemic, and can limit the possibility of formation heart failure.Can expect that the treatment that reduces myocardial infarction can save the life of high-risk sufferer, shorten the hospital stays, improve the quality of living and reduce total health care spending.
Yet unfortunately, also there is not such methods of treatment at present.Though once attempted various methods of treatment but all do not had successfully.(referring to the survey article of Downey and Cohen, Prog CardiovascDis (cardiovascular diseases progress), 48:363-371,2006).Antithrombotic is intervened, as acetylsalicylic acid, clopidogrel and ReoPro
At present recommendedly be used to prevent obturation coronarius/inaccessible again.Yet they can not provide direct protection to reperfusion injury.In fact, acetylsalicylic acid can hinder the endogenous myocardial approach, and increases generation people such as (, J Pharmacol Exp Ther, 310:185-191,2004) Gross of infraction.
Summary of the invention
The invention provides and a kind ofly suppress the reperfusion injury of sufferer and/or the method for infraction by using platelet glycoprotein VI (GPVI) inhibitor, wherein, described platelet glycoprotein VI (GPVI) is the main collagen protein acceptor that is present in platelet surface.The present invention also provides described inhibitor to be used for the treatment of purposes in the medicine of reperfusion injury and/or infraction in preparation.
GPVI be expressed on thrombocyte and the megalokaryocyte single-mindedly and and the collagen protein combination, described collagen protein is the easiest thrombosed stroma protein under blood vessel endothelium.The breaking of atherosclerotic plaque, ischemic and reperfusion injury can make collagen protein be exposed to comprise hematoblastic blood ingredient.The combination of thrombocyte GPVI and collagen protein plays a crucial role at the adhesion of damaged vascular site and platelet activation subsequently and gathering to thrombocyte.Thrombocyte GPVI inhibitor stops thrombocyte GPVI and is present in interaction between the collagen protein of vessel wall.Once showed the hematoblastic activation of reduction in the past though suppress GPVI, surprisingly, the present invention shows that suppressing GPVI also has the effect of direct protection heart, can be used to suppress reperfusion injury and/or infraction.
Described thrombocyte GPVI inhibitor can be antibody, protein fragments or micromolecular compound.Described antibody includes, but not limited to monoclonal anti-GPVI antibody.Monoclonal antibody comprises the active antibody fragment.The active antibody fragment can be Fab fragment, the F (ab) of chemistry, enzymolysis or reorganization preparation
2Fragment or comprise at least one peptide to GPVI polypeptide, peptide or its specific complementarity-determining region of natural variant tool (CDR).Exemplary antibody comprises monoclonal antibody OM1, OM2, OM3 and OM4 and their humanization variant or its active fragments of Muridae.Peptide fragment includes, but not limited to the collagen protein calmodulin binding domain CaM of GPVI and soluble g PVI.
Description of drawings
Figure 1 shows that wild-type mice and GPVI knock out the comparison of mouse myocardial infarction size.At 30 minutes ischemics with after pouring into again in 24 hours, to compare with wild-type mice, the myocardial infarction size that GPVI knocks out mouse obviously diminishes.Each empty circles is represented the infraction size from individual mouse, and the circle of blacking is represented cell mean ± SD.With the t check data are analyzed, and thought that p＜0.05 has statistical significance.
Fig. 2 a is depicted as at ischemic with again after the perfusion, wild-type mice and GPVI is knocked out the comparison of P-selectin expression in the cardiac muscle of mouse.The typical fluoroscopic image that has shown endocardium and middle level cardiac muscle.The expression decreased (dim background fluorescence is because the autofluorescence of cardiac muscle causes) that in GPVI knocks out the mouse cardiac muscle, is the P-selectin of bright green (or in the picture of black and white version, being brilliant white).The heart that similar result also knocks out mouse from 5 routine wild-type mices and 5 routine GPVI respectively obtains.Fig. 2 b shown to the zone that the P-selectin high expression level is arranged quantitatively.The level that GPVI knocks out the P-selectin of (KO) mouse is starkly lower than wild-type (WT) mouse (n=5), this show GPVI bring out the cardiac muscle in platelet activation and the gathering in play an important role.
Figure 3 shows that wild-type mice exposes at the collagen protein that occurs in its heart owing to pour into again behind the ischemic.Left figure shows typical case's section of then carrying out the heart after 15 minutes and reperfusion through 30 minutes ischemic.The collagen protein (dim background fluorescence is because the autofluorescence of cardiac muscle causes) that bright green (or being brilliant white in the picture of black and white version) expression comes out.Also obtained similar result from other 3 animals.Right figure shows the typical case's section that does not have follow-up dabbling heart again through 30 minutes ischemic.Green fluorescence (or not having brilliant white in the picture of black and white version) do not occur, this shows does not have collagen protein to expose.Also obtained similar result from other 2 animals.In sum, these data show endothelial injury are taking place between flush phase again.
Fig. 4 a has confirmed the effect that the infraction of anti--GPVI antibody OM2 in the monkey body reduces.The figure illustrates the scatter diagram of risk band (risk zone) and infarct, wherein draw the tropic for each treatment group of indicating.Infraction is linear dependence with the size of risk band in the control group monkey.Because all numerical points are below the tropic of control group (p＜0.05) all, so no matter give dose or two doses, the monkey of OM2 treatment has all reduced myocardial infarction.And to reduce be similar to infraction in the monkey of dose or two doses treatment, this show again between flush phase OM2 brought into play provide protection.(ANOVA) analyzes the infraction data by variance analysis.Fig. 4 b has confirmed the restraining effect of OM2 to the platelet aggregation in the monkey blood.Use OM2 (2mg/kg) before (before the administration) and afterwards (after the administration 4 hours) in the monkey body, take a blood sample.In the experiment of adopting living materials, measure the platelet aggregation that collagen protein brings out with the whole blood aggregometer.Fig. 4 b shows that the typical case of the platelet aggregation that collagen protein brings out measures numerical value.In the whole blood of the animal of accepting OM2, the platelet aggregation that collagen protein brings out is suppressed fully.
Embodiment
" infraction " typically refers to the tissue necrosis that the upstream occlusion owing to its arterial blood confession causes.Lacking oxygen containing blood can make cell downright bad because of lacking nutrition.Infraction can any organ of influence, but in that these need in high-energy and the hypermetabolism active tissue to understand and more often and quicker take place as heart (less than 20-120 minute).
Term " myocardial infarction " is meant in this application owing to the coronary flow that flows to myocardial region reduces the myocardial necrosis that causes suddenly.Cardiac muscle only can bear very short time the ischemic of (less than 5 minutes) and unlikely the damage.Blood flow can not recover generally can produce the reversible damage in 5-20 minute.The ischemic of longer time can produce nonvolatil damage, i.e. necrocytosis/necrosis/infarct usually.Because the regenerative power of cardiac muscle is very limited, the loss of muscle can be nonvolatil." endothelial function imbalance " is meant owing to ischemic and pours into the endothelium necrosis that causes again or the forfeiture of normal function.
After " reperfusion injury " is meant the ischemic of for some time, when blood for coming back to caused tissue injury when organizing.Come the oxygen of autoblood and the disappearance of nutritive ingredient can cause such situation: the round-robin recovery can cause the inflammation of being brought out by oxidative stress and the recovery of oxidative damage rather than normal function." reperfusion injury of cardiac muscle " is meant the reperfusion injury that occurs in cardiac muscle, and " endothelium reperfusion injury " is meant the reperfusion injury that occurs in endothelium.
" sufferer " is meant anyone or the inhuman animal that needs treatment to reduce generation, possibility or the degree of infraction and/or reperfusion injury in this application." sufferer " also comprises the individuality that those once suffered heart attack or the heart attack risk is arranged, it comprises, but be not limited to, those have been diagnosed as the people of cardiovascular disorder, for example the individuality of coronary artery disease (CAD), systemic hypertension, bicuspid aortic valve disease, hypertrophic cardiomyopathy or mitral leaflet prolapse; Those or are once being had a heart attack and/or the individuality of in heart failure (comprising congestive heart failure (CHF)); And those need the individuality that carries out the selectivity heart operation of temporary interruption coronary flow (for example during bypass surgery).The inhuman animal for the treatment of comprises that all are raised and train or wild vertebrates, includes, but not limited to mouse, rat, rabbit, fish, bird, hamster, dog, cat, pig, sheep, horse, ox and inhuman primate.
Term " inhibition " is meant the minimizing of any phenotypic characteristic or stops, perhaps being meant described feature generation, degree or possibility minimizing or stop.Therefore, " suppress reperfusion injury and/or infraction " be meant reperfusion injury and/or block measurable minimizing or stop.
" thrombocyte GPVI inhibitor " is meant any antibody, protein fragments or micromolecular compound that can suppress thrombocyte GPVI function.The function of thrombocyte GPVI comprise thrombocyte GPVI and the collagen protein for example in vessel wall, found between interaction.Other function comprise platelet aggregation that collagen protein brings out, platelet adhesion reaction to the fixed collagen protein, the thromboxan A that brings out of the Triphosaden secretion brought out of collagen protein and collagen protein
2Formation.
Term " antibody " is known in the art, and it comprises monoclonal antibody.Monoclonal antibody of the present invention comprises the active antibody fragment, as Fab fragment, the F (ab) by chemistry, enzymolysis or reorganization preparation
2Fragment or comprise at least one peptide segment to GPVI polypeptide, peptide or its specific complementarity-determining region of natural variant tool (CDR).If anti--GPVI antibody combines with GPVI polypeptide, peptide or its natural variant, its dissociation constant (Kd) is equal to or less than 10
-7M, the combination between them is exactly " specific combination ".In yet another embodiment of the present invention, anti--GPVI antibody and GPVI polypeptide, peptide or its natural variant are equal to or less than 10 with Kd
-8M is combination specifically.In another embodiment of the present invention, of the present invention resisting-GPVI antibody and polypeptide, peptide or its natural variant are equal to or less than 10 with Kd
-9M is combination specifically.As segmental saturated in conjunction with thermoisopleth by measuring with the IgG of 125I mark or its, or by replacing with unlabelled IgG homology
125The IgG of I mark and adopt nonlinear regression analysis (as by Motusky at Analyzing Datawith GraphPad Prism (1999), GraphPad Software Inc., San Diego puts down in writing among the CA) can be at an easy rate measure avidity in conjunction with object or antibody by the technology of routine.Other technology also is known in the art, for example, by people such as Scatchard at Ann.NY Acad.Sci., those methods of describing among the 51:660 (1949).
U.S. Patent Application Publication No.2007/0207155 describes the preparation of monoclonal antibody and humanized antibody thereof in detail.U.S. Patent Application Publication No.2007/0207155 has also put down in writing monoclonal antibody OM1, OM2, OM3 and the OM4 with above-mentioned binding characteristic, and comprises at least one peptide segment to GPVI polypeptide, peptide or its specific complementarity-determining region of natural variant tool (CDR).And, U.S. Patent No. 6,998,469 and U.S. Patent Application Publication No.2007/0207155 in GPVI polypeptide, peptide or its natural variant have all been described, incorporate the both into the application in the complete mode of quoting.
" micromolecular compound " is meant organic non-protein compound, and its size is 1500Da to the maximum.But micromolecular compound synthetic or derive by natural product extract.Its crucial constitutional features is an inflexible polycyclic core texture normally, and this kind structure has reduced the entropy (entropic cost) that expends because of small molecules and protein bound.Micromolecular compound of the present invention suppresses the function of thrombocyte GPVI, includes but not limited to the interaction of thrombocyte GPVI and collagen protein.
As mentioned above, " peptide fragment " comprises and comprises at least one peptide segment to GPVI polypeptide, peptide or the specific CDR of its natural variant tool, and its example is documented among the U.S. Patent Application Publication No.2007/0207155.Other peptide fragment can comprise the collagen protein calmodulin binding domain CaM of GPVI.Complete GPVI sequence has been disclosed in people such as Chemetson, J.Biol.Chem.274:29019-24 (1999); WO 00/68377; People such as Jandrot-Perrus, Blood 96:1798-807 (2000); With people such as Ezumi, among the Biochem BiophysRes Commun.277:27-36 (2000), and drawn GPVI go up main collagen protein mating surface (O ' people such as Connor, J.Biol.Chem.281 (44): 33505-10 (2006); People such as Dumont, J.Mol.Biol.361 (5): 877-87 (2006); People such as Horii, Blood 108 (3): 936-42 (2006); People such as O ' Connor, J.Chromb Haemost.4 (4 :) 869-73 (2006)).In addition, the GPVI of solubility (sGPVI) (zone, extracellular that comprises GPVI) also demonstrates the combination that can suppress GPVI and collagen protein, therefore can suppress the platelet aggregation that collagen protein brings out people such as (, Blood96:1798-807 (2000)) Jandrot-Perrus.
Treatment to sufferer comprises the thrombocyte GPVI inhibitor of using pharmacologically effective dose.Those of ordinary skill in the art can determine to use the optimal dose and the dosage schedule of these inhibitor by rule of thumb.Yet pharmacologically effective dose is the amount of reperfusion injury, infraction or the ischemic event that can suppress sufferer.
In the whole course of treatment, can use pharmacologically effective dose with dose or multidose.Inhibitor of the present invention can be used by any way that those skilled in the art were familiar with, for example, and vein (IV) bolus injection, continuous infusion or intermittent infusion.In other embodiment, described inhibitor can be by in the intraperitoneal (IP), body, in the intraarticular, ventricle, in the sheath, in the intramuscular, subcutaneous, local, tonsilla, mucous membrane, nose, use through skin, intravaginal, oral or suction.
By following embodiment the present invention is illustrated, yet these embodiment do not limit the present invention in any way.
Embodiment 1
The GPVI disappearance is to the protection of mouse heart
With the isofluranum of 1-1.5% wild-type mice of the same age and GPVI are knocked out mouse and anaesthetize,, and it is attached on the pressure controlled respirator by the endotracheal tube intubate.Ventilate to animal with room air and 100% oxygen (by 4: 1 volume ratios).Before undergoing surgery, give mouse gentamicin (0.7mg/kg IM).In whole experiment, carefully monitor body temperature and body temperature is remained on 37-37.5 ℃ with hot-plate or heating lamp by the rectal bougie that is connected with digital thermometer.In research in advance, conduit is inserted carotid artery with the blood pressure that the detects mouse promoting circulation of blood gas analysis of going forward side by side.Be that mouse can keep physiological hemodynamic in these experimental procedures in order to guarantee to adopt like this.
By dissecting microscope, open the thoracic cavity by left thoracotomy.With the taper pin with 8-0 nylon suture (Ethicon, Inc.Johnson ﹠amp; Johnson Co.Somerville NJ) passes apart from left auricle of heart top 2-3mm from the left anterior descending coronary artery below, and the end of suture passes a plastics tubing.Make the coronary vasodilator obturation with respect to plastics tubing pulling suture.At ischemic and again between the paracmasis after the perfusion, coronary vasodilator inaccessible and dabbling more successfully be to confirm by range estimation (promptly by observing the pale asphyxia and because the shiny red that hyperemia recovers after the venting of far-end cardiac muscle when the pulling suture).Ischemic continues 30 minutes.After unblocking, successively close the thoracic cavity with suture.The recheton (Ketofen) of a dosage of injection (2.5mg/kg, IM).After recovering autonomous respiration, mouse is removed and is placed into temperature and humidity control and be rich in the equipment of oxygen from ventilator.After mouse obtains the normal attitude ability, it is put back in the cage spend 24 hours.
When experiment finishes (second day), use heparin (1U/g IP) to mouse, use vetanarcol (100mg/kg IP) anesthesia subsequently.The excision heart also adopts Lang Genduofu device (Langendorf apparatus) to pour into Krebs-Henseleit solution by aortic cannulation (23-gauge needle).The occlusion areas of drawing is to pour into the profile of zone (risk zones) more then, formerly coronary artery is tightened and with 1% fluorescent grain (diameter 1-10 μ m in Bi Sai position, Duke Scientific, Palo Alto, normal saline solution perfusion aortic root (1mL in 3 minutes) CA).The result of this operation is, by the left ventricle (LV) of the coronary artery supply of previous obturation partly (risk zones) determined under UV-light owing to there is not fluorescence, and the rest part of left ventricle is dyed mazarine.With freezing 20 minutes of heart, be cut into 5-7 sheet transverse section then.In order to present the profile of infarct and survival myocardium, the heart section was cultivated 20 minutes with 1% triphenyltetrazolium chloride (TTC) in phosphate buffered saline buffer (pH7.4,37 ℃).Section is fixing in 10% neutral buffered formaldehyde, take pictures after 24 hours.The border that shows infraction, ischemia-reperfusion (risk zones) and no ischemic zone.Measure corresponding area with the computerize planimetry, and calculate the per-cent of the size of infraction as risk zones by these take off data.
Wild-type mice and GPVI knock out that the risk zones size of mouse is similar (to be respectively 0.020 ± 0.004cm
3With 0.022 ± 0.005cm
3).45 ± 18% of the infarct area of wild-type mice (infraction size) average out to risk zones.GPVI knocks out mouse infarct area (infraction size) and obviously diminishes 22 ± 8% of average out to risk zones.These data are summarised among Fig. 1.
GPVI knocks out the reduction that P-selectin is expressed in the mouse cardiac muscle
Hematoblastic activation is to measure by the expression of P-selectin, and described P-selectin is stored in thrombocyte α-particle, in case be activated, can transfer to hematoblastic surface soon.Adopt immunohistology to measure the expression of P-selectin.
Mouse intracorporeal heart ischemia/reperfusion: as described in embodiment 1, carry out the mouse heart ischemia/reperfusion.GPVI knocks out with wild-type mice and accepted left anterior descending coronary artery (LAD) inaccessible 30 minutes, and then pours into 15 minutes.After 15 minutes and reperfusion of LAD, take out heart and clean with DPBS, be cut into two parts by minor axis then and put into 4% paraformaldehyde immediately and the 0.1M phosphate buffered saline buffer with fixing organization.After 2 hours, this tissue transferred in 25% sucrose spend the night.
The immunofluorescence assay of P-selectin:, heart tissue is cut into the transverse section of 20 μ m and makes its dry about 30 minutes at second day.Section on each slide glass is made the annulus mark with the PAP pen, and make its dry about 10 minutes.Fine laundering slide glass in 0.01M PBS-0.1%Triton (PBST), and (10%NDS PBST) cultivated 30-60 minute at room temperature to use normal donkey serum.With rabbit anti--mouse platelets selects protein polyclone antibody (Chemicon International) that P-selectin is detected, and with FITC-anti--rabbit igg (Jackson ImmunoResearch lab) develops.
Fluoroscopic image: adopt Zeiss Laser Scanning Confocal Microscope (LSM510) or conventional fluorescent microscope to obtain fluoroscopic image.Measure at the 480nm fluorescence excitation and at 540nm.Ischemic 30 minutes with after pouring into 15 minutes again, (Fig. 2 a) to detect high-caliber P-selectin in the cardiac muscle (endocardium and middle level cardiac muscle) of wild-type mice.Detecting quite few P-selectin in GPVI knocks out the cardiac muscle of mouse expresses.For the quantitative expression level, be determined at the area size that strong green fluorescence is arranged in the ischemic area.Data presentation is compared with wild-type mice, and the total area size that GPVI knocks out the interior P-selectin expression of the heart zone of mouse obviously reduces (Fig. 2 b).
The endothelium reperfusion injury
In having the normal heart of healthy vascular system, the collagen protein of endothelium prevention closely in the extracellular matrix of vessel wall contacts with the blood circulation composition.If endothelium is damaged, for example at ischemic with again between flush phase, collagen protein may come out.Because GPVI optionally combines with collagen protein, GPVI is used to detect intravital endothelium reperfusion injury.For this purpose, use fluorescent marker FITC mark recombinant chou sGPVI (sGPVI-FITC), and the sGPVI-FITC intravenous injection is arrived in the mouse body.The sGPVI-FITC that is injected into combines with the collagen protein that exposes owing to endothelial injury.SGPVI-FITC with expose collagen protein bonded level and can under fluorescent microscope, carry out Histological determining, and provide measuring method for endothelial injury.
Took place preceding 10 minutes at wild-type rat heart ischemic (30 minutes), sGPVI-FITC (2mg/kg) is expelled in its body.In some animals, pour into (15 minutes) behind the ischemic more therein.Through observing the visible marking (Fig. 3) of vascular system in the dabbling heart again, this shows the obvious damage of endothelium, therefore shows that collagen protein is exposed to the round-robin blood ingredient.On the contrary, not through in the dabbling heart again, do not observe vascular system (Fig. 3) with the sGPVI-FITC mark.The perfusion again of these data presentation Ischemic Heart tissues causes endothelial injury.
The cardioprotection of anti--GPVI antibody
Use from the weight of China macaque as 2.0-2.5kg.The monkey overnight fasting that experiment is selected for use is also used ketamine (10mg/kg 1M) calmness.Injecting atropine (0.05mg/kg 1M) more in addition.An intravenous catheter is inserted into the shank vein.Finish anesthesia and in experimentation, use boost with vetanarcol (10-15mg/kg IV).Expose tracheae by the center cervical incision, and insert tracheal catheter.By small-sized animal respirator and 40%O
2/ 60%N
2Mixed gas be animal ventilation.One conduit is inserted carotid artery to measure blood pressure and to collect the artery blood sample.In the 4th intercostal, carry out left thoracotomy and expose heart.Left anterior descending coronary artery can see once in a while, but usually since the fat of covering be blocked.To be through 2-0 suture on the pin blind passing below the blood vessel bundle in interventricular groove, and as far as possible near the starting point of artery.The end of suture passes a very short polyethylene catheter to form a snare.The success of snare confirms by following phenomenon: when being tightened up for 10 seconds, snare observes the cyanosis of heart antetheca and stopping of contraction, and the hyperemia of tissue and the recovery of contraction when snare is decontroled.A conduit is inserted left auricle of heart be used for the microballoon injection.Link ECG and lead logotype in measuring heart rate and QRS form.With hot-plate monkey is heated and to make the rectal temperature that records reach 38 ℃.
Finish operation preparation work and balance after at least 20 minutes, record baseline heart rate, blood pressure and ECG, and with coronary occlusion 90 minutes.Continuous monitoring ECG, heart rate and blood pressure, and the per minute record once write down once in the time of 10 minutes in 5 minutes, record once finished up to obturation in after this per 10 minutes.When the inaccessible phase finishes, discharge snare and make coronary artery reperfusion.Equally, continuous monitoring ECG, heart rate and blood pressure, and the per minute record once write down once in the time of 10 minutes in 5 minutes, the once flush phase end again up to 4 hours of record in after this per 10 minutes.If the generation ventricular fibrillation uses electrical defibrillator to make the rhythm and pace of moving things be transformed into hole.
After 4 hours the perfusion again, take out heart and it is hung on the perfusion instrument by aortic root.From the blood of coronary artery and heart, once more after the obturation, (Microgenics Corp., Freemont CA) add perfusion liquid to retroperfusion salt solution with 2-9 μ m green fluorescent microspheres at coronary artery with flushing.Like this, fluorescent microsphere only enters the cardiac muscle by open coronary perfusion, and risk zones (or risk band) is divided out as the myocardial region that does not contain fluorescent microsphere.Heart is placed on the dry ice freezing, is cut into the section of 2-3mm perpendicular to its major axis.To cut into slices at 1% triphenyltetrazolium chloride (the TTC) (GFSChemicals that is heated to 37 ℃, Powell, OH) cultivated 8-10 minute in, put into 10% formalin then and organize anticorrosion and improve and dyeed by TCC and not by the colour contrast between the painted tissue of TCC.The normal perfused tissue that TTC will contain intact NADH is dyed brick-red, however the blocking tissue that has wherein discharged and washed off cofactor be not colored and be white in color, perhaps owing to myocardium internal hemorrhage is black.Section is pressed between the plastic plate of the 2mm of just in time being separated by.The size of the risk region that will discern under UV-light and the infarct area of discerning under white light is depicted on the plastic wrap.Measure area and come volume calculated with planimetry by area be multiply by 2mm.
Control animals is carried out coronary occlusion (90 minutes) and perfusion (4 hours) again under the situation of not using anti--GPVI antibody.In an anti--GPVI antibody treatment group, animals received the OM2Fab fragment of two doses (monoclonal mouse Anti-Human GPVI antibody; Referring to people such as Matsumoto, Thromb Res, 119:319-329,2007; With U.S. Patent Application Publication No.2007/0207155) (each 2mg/kg), dosage was used before 10 minutes at ischemic for the first time, and dosage is just in time being used before the perfusion more for the second time.Because immunofluorescence data (referring to Fig. 1-3) have supported GPVI in role between flush phase again, so in second anti--GPVI antibody treatment group, animal has just in time been accepted the OM2 Fab fragment (2mg/kg) of dose before perfusion again.By ANOVA analysis of cardiac infraction, p value＜0.05 is considered to have statistical significance.
Being of a size of X-coordinate with the risk band draws the figure of infraction size rather than it is depicted as chart of percentage comparison.This is because do not find the source of chart of the infraction size/risk mark cun of control animals, for rodent such problem people such as (, Am J Physiol, 267:H2383-H2390,1994) Ytrehus is arranged also.As people such as Flameng (Basic Res Cardio 185:392-403,1990) pointed to baboon like that, the size of risk band also is an important determinative of infraction size in macaque.Therefore, when the size of risk band hour, even without any intervention, also the size of measurable infraction can be little.And when the size of risk band less than 0.6cm
3The time, even in the control group monkey, also can expect and not block.When being of a size of X-coordinate with the risk band and drawing the figure of infraction size, because the tropic moves right, OM2 antibody (no matter two doses or dose) demonstrates tangible cardioprotection.This moving shows that the monkey that OM2 handles has littler infraction size under same risk mark is very little.With twice similar with degree of protection in the monkey that dose OM2 handles, this and following identical of views: the interaction of the thrombocyte-collagen protein by GPVI is brought out reperfusion injury and is provided Cardioprotective to this interactional inhibition.
Whether suppressed hematoblastic activation on one's body in order to study OM2, before and after using OM2, taken a blood sample these monkeys.Use whole blood aggregometer (Chrono-log, Corporation PA) to measure the platelet aggregation that external collagen protein brings out then.With 1: 1 (v/v) dilute blood of salt solution, and by collagen protein (0.5 μ g/mL; Horm, Nycomed, Germany) bring out begin to assemble before, earlier it was cultivated 5-10 minute in aggregometer under 37 ℃.Along with (Aggro/link v4.75 Chrono-log) assembled in the increase monitoring of electrode impedance in blood sample.
The typical observed value of the platelet aggregation that brings out of collagen protein in the blood sample of (before the administration) and (after the administration) collection after 4 hours before Fig. 4 b is presented at OM2 and uses.These data show that the OM2 (2mg/kg) that before pouring into again monkey is used has suppressed the platelet aggregation that collagen protein brings out (measuring by the analyzed in vitro method) fully.The OM2 that uses with 0.4mg/kg has shown similar restraining effect (data not shown).
Can separate specification sheets by more thorough geography with reference to the document of quoting in this specification sheets, with these documents with the complete the application that incorporates into of way of reference.According to explanation of the present invention disclosed here and enforcement, other embodiments of the present invention it will be apparent to those skilled in the art that.Think that this specification sheets and embodiment only are in order to explain and illustrate that the claims of enclosing have shown real essence of the present invention and scope.
Claims (30)
1. one kind comprises the method that platelet glycoprotein VI (GPVI) inhibitor need suppresses by it sufferer reperfusion injury and/or infraction of using, and wherein, described inhibitor suppresses the interaction between thrombocyte GPVI and the collagen protein.
2. method according to claim 1, wherein, described reperfusion injury and/or infraction are reperfusion injury of cardiac muscle and/or myocardial infarction.
3. method according to claim 1, wherein, described reperfusion injury and/or infraction are endothelium reperfusion injury and/or endothelial function imbalance.
4. method according to claim 1, wherein, described sufferer once had heart attack.
5. method according to claim 1, wherein, described sufferer need cause the elective surgery of coronary flow temporary interruption.
6. method according to claim 1, wherein, described inhibitor is to GPVI polypeptide, peptide or the specific antibody of its natural variant tool.
7. method according to claim 6, wherein, described antibody is selected among OM1, OM2, OM3 and the OM4.
8. method according to claim 6, wherein, described antibody is OM2.
9. method according to claim 6, wherein, described antibody is by humanization.
10. method according to claim 6, wherein, described antibody is the active antibody segment, it is selected from Fab segment, the F (ab) of chemistry, enzymolysis or reorganization preparation
2Segment or comprise at least one peptide segment to GPVI polypeptide, peptide or its specific complementarity-determining region of natural variant tool (CDR).
11. method according to claim 10, wherein, described active antibody segment is the antibody fragment that is selected among OM1, OM2, OM3 and the OM4.
12. method according to claim 11, wherein, described antibody is OM2.
13. method according to claim 10, wherein, described active antibody segment is by humanization.
14. method according to claim 1, wherein, described inhibitor is the peptide segment of GPVI.
15. method according to claim 14, wherein, the peptide segment of described GPVI is the collagen protein calmodulin binding domain CaM of GPVI.
16. method according to claim 14, wherein, described peptide segment is soluble g PVI (sGPVI).
17. platelet glycoprotein VI (GPVI) inhibitor is in the purposes that is used for preparing the medicine for the treatment of reperfusion injury and/or infraction, wherein, described inhibitor suppresses the interaction between thrombocyte GPVI and the collagen protein.
18. purposes according to claim 17, wherein, described reperfusion injury and/or infraction are reperfusion injury of cardiac muscle and/or myocardial infarction.
19. purposes according to claim 17, wherein, described reperfusion injury and/or infraction are endothelium reperfusion injury and/or endothelial function imbalance.
20. purposes according to claim 17, wherein, described inhibitor is to GPVI polypeptide, peptide or the specific antibody of its natural variant tool.
21. purposes according to claim 20, wherein, described antibody is selected among OM1, OM2, OM3 and the OM4.
22. purposes according to claim 21, wherein, described antibody is OM2.
23. purposes according to claim 20, wherein, described antibody is by humanization.
24. purposes according to claim 20, wherein, described antibody is the active antibody segment, and it is selected from Fab segment, the F (ab) of chemistry, enzymolysis or reorganization preparation
2Segment or comprise at least one peptide segment to GPVI polypeptide, peptide or its natural variant tool specific complementarity-determining region territory (CDR).
25. purposes according to claim 24, wherein, described active antibody segment is for being selected from antibody fragment among OM1, OM2, OM3 and the OM4.
26. purposes according to claim 25, wherein, described antibody is OM2.
27. purposes according to claim 24, wherein, described active antibody segment is by humanization.
28. purposes according to claim 17, wherein, described inhibitor is the peptide segment of GPVI.
29. purposes according to claim 28, wherein, the peptide segment of described GPVI is the collagen protein calmodulin binding domain CaM of GPVI.
30. purposes according to claim 28, wherein, described peptide segment is soluble g PVI (sGPVI).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US98433407P | 2007-10-31 | 2007-10-31 | |
| US60/984,334 | 2007-10-31 | ||
| PCT/US2008/012311 WO2009058326A1 (en) | 2007-10-31 | 2008-10-29 | Uses of a glycoprotein vi (gpvi) inhibitor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101874107A true CN101874107A (en) | 2010-10-27 |
Family
ID=40591365
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200880114053A Pending CN101874107A (en) | 2007-10-31 | 2008-10-29 | The purposes of glycoprotein VI (GPVI) inhibitor |
Country Status (16)
| Country | Link |
|---|---|
| US (1) | US20100297116A1 (en) |
| EP (1) | EP2212416A4 (en) |
| JP (1) | JP2011502123A (en) |
| KR (1) | KR20100075585A (en) |
| CN (1) | CN101874107A (en) |
| AR (1) | AR069120A1 (en) |
| AU (1) | AU2008319336A1 (en) |
| BR (1) | BRPI0818807A2 (en) |
| CA (1) | CA2703770A1 (en) |
| IL (1) | IL205453A0 (en) |
| MX (1) | MX2010004537A (en) |
| RU (1) | RU2010121878A (en) |
| SG (1) | SG185307A1 (en) |
| TW (1) | TW200936606A (en) |
| WO (1) | WO2009058326A1 (en) |
| ZA (1) | ZA201002990B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2336188A1 (en) * | 2009-12-18 | 2011-06-22 | Sanofi-Aventis | Novel antagonist antibodies and their Fab fragments against GPVI and uses thereof |
| EP2933269A1 (en) | 2009-12-18 | 2015-10-21 | Sanofi | Novel antagonist antibodies and their fab fragments against gpvi and uses thereof |
| EP2397495A1 (en) * | 2010-06-21 | 2011-12-21 | Sanofi | Novel antagonist antibodies and their Fab fragments against GPVI and uses thereof |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7928066B1 (en) * | 1999-05-07 | 2011-04-19 | Aventis Pharma Deutschland Gmbh | Recombinant platelet collagen receptor glycoprotein vi and its pharmaceutical use |
| US20040001826A1 (en) * | 1999-06-30 | 2004-01-01 | Millennium Pharmaceuticals, Inc. | Glycoprotein VI and uses thereof |
| AU7097400A (en) * | 1999-09-01 | 2001-03-26 | Otsuka Pharmaceutical Co., Ltd. | Platelet membrane glycoprotein vi (gpvi) dna and protein sequences, and uses thereof |
| EP1224942A1 (en) * | 2001-01-23 | 2002-07-24 | Bernhard Dr. Nieswandt | Use of JAQ1 (monoclonal antibody anti GPVI) as a medicament for the protection against thrombotic diseases |
| GB0130543D0 (en) * | 2001-12-20 | 2002-02-06 | Univ Cambridge Tech | Human antibodies and their use |
| EP1369128A1 (en) * | 2002-06-07 | 2003-12-10 | Procorde GmbH | Inhibitors of glycoprotein VI and their therapeutic use |
| WO2005007800A2 (en) * | 2003-07-18 | 2005-01-27 | Mochida Pharm Co Ltd | Monoclonal antibody against platelet membrane glycoprotein vi |
| US7645592B2 (en) * | 2004-04-29 | 2010-01-12 | Otsuka Pharmaceutical Co., Ltd. | Glycoprotein VI antibodies and methods of use thereof |
| WO2005111083A2 (en) * | 2004-04-29 | 2005-11-24 | Otsuka Pharmaceutical Co., Ltd. | Antibodies specific for glycoprotein vi and methods of producing these antibodies |
| CA2606450A1 (en) * | 2005-04-28 | 2006-11-09 | Mochida Pharmaceutical Co., Ltd. | Anti-platelet membrane glycoprotein vi monoclonal antibody |
-
2008
- 2008-10-29 AU AU2008319336A patent/AU2008319336A1/en not_active Abandoned
- 2008-10-29 SG SG2012076709A patent/SG185307A1/en unknown
- 2008-10-29 CN CN200880114053A patent/CN101874107A/en active Pending
- 2008-10-29 EP EP08843804A patent/EP2212416A4/en not_active Withdrawn
- 2008-10-29 JP JP2010531074A patent/JP2011502123A/en active Pending
- 2008-10-29 KR KR1020107009550A patent/KR20100075585A/en not_active Application Discontinuation
- 2008-10-29 BR BRPI0818807-6A2A patent/BRPI0818807A2/en not_active IP Right Cessation
- 2008-10-29 US US12/740,620 patent/US20100297116A1/en not_active Abandoned
- 2008-10-29 RU RU2010121878/15A patent/RU2010121878A/en not_active Application Discontinuation
- 2008-10-29 CA CA2703770A patent/CA2703770A1/en not_active Abandoned
- 2008-10-29 MX MX2010004537A patent/MX2010004537A/en active IP Right Grant
- 2008-10-29 WO PCT/US2008/012311 patent/WO2009058326A1/en active Application Filing
- 2008-10-30 AR ARP080104753A patent/AR069120A1/en not_active Application Discontinuation
- 2008-10-31 TW TW097142069A patent/TW200936606A/en unknown
-
2010
- 2010-04-29 ZA ZA2010/02990A patent/ZA201002990B/en unknown
- 2010-04-29 IL IL205453A patent/IL205453A0/en unknown
Non-Patent Citations (1)
| Title |
|---|
| JANDROT-PERRUS M ET AL: "Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily", 《BLOOD》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| IL205453A0 (en) | 2010-12-30 |
| US20100297116A1 (en) | 2010-11-25 |
| JP2011502123A (en) | 2011-01-20 |
| CA2703770A1 (en) | 2009-05-07 |
| BRPI0818807A2 (en) | 2014-10-29 |
| RU2010121878A (en) | 2011-12-10 |
| AU2008319336A1 (en) | 2009-05-07 |
| TW200936606A (en) | 2009-09-01 |
| EP2212416A1 (en) | 2010-08-04 |
| SG185307A1 (en) | 2012-11-29 |
| EP2212416A4 (en) | 2013-01-09 |
| ZA201002990B (en) | 2011-07-27 |
| AR069120A1 (en) | 2009-12-30 |
| WO2009058326A1 (en) | 2009-05-07 |
| KR20100075585A (en) | 2010-07-02 |
| MX2010004537A (en) | 2010-05-20 |
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